Method for preparing tetracycline ii



United States Patent METHOD FOR PREPARING TETRACYCLINE lI MasaoArishima, Nakano-ku, and Yasuharu Sekizawa, Shibuya-ku, Tokyo, Japan,assignors to American Cyanamid Company, New York, N.Y., a corporation ofMaine No Drawing. Filed June 4, 1956, Ser. No. 588,962

8 Claims. Cl. 195-80) This invention relates to the manufacture oftetracycline'and more particularly to the production of tetracycline bymicroorganisms which also tend to form chlortetracycline in fermentationmedia containing chlorides.

Recently it has been discovered that microorganisms of the genusStreptomyces which produce chlortetracycline will also producetetracycline, particularly if the chloride ion concentration of thefermentation medium is kept low. This can be done by providingfermentation media from which chloride ions are excluded, either bymaking up the fermentation medium with chloride-free components or bytreating the medium with agents which remove or sequester the chlorideions, thus making them unavailable for the formation ofchlortetracycline.

Unfortunately some of the most eifective components of fermentationmedia for the production of the tetracycline antibiotics containsubstantial quantities of chloride ions. Corn steep liquor is one of themost effective nutrient substances for the production of thetetracycline antibiotics as well as many other antibiotics. Apparently,this natural material contains something that is especially desired bythe fermenting microorganism. Highest yields of antibiotic are thereforeobtained when a portion of corn steep liquor is included in the aqueousnutrient medium. Corn steep liquor contains substantial amounts ofchloride ions, however, and if formation of chlortetracycline is to bekept at reasonably low levels, it has heretofore been considerednecessary to reduce the chloride content of this material. The sameapplies to a number of other naturally occurring nutrient materialswhich are advantageously used in the fermentation process.

Several means of reducing the chloride ion content of aqueous nutrientsolutions for tetracycline production have been suggested. Precipitationof the chloride ion as silver chloride is a very effective means ofreducing the chloride ion content. However, this is an expensive processrequiring the use of expensive raw materials, special equipment, and atoxic gas, hydrogen sulfide, for recovery of silver.

Ion exchange resins have also been suggested to reduce the chloride ioncontent of fermentation media. These substances tend to remove somesalts and organic substances of unidentified composition which aredesirable components of the fermentation media. As a result, low yieldsof antibiotic may be obtained when using nutrient solutions which havebeen pretreated with ion exchange resins to remove chloride ions.

The chloride deprivation systems still leave much to be desired,however, because of the capital investment required, the relativelycomplicated means of removing chloride ions from the fermentationmedium, as Well as the fact that the fermentation medium can in suchinstances be composed only of those raw materials from which chlorideions can be easily removed or which are naturally low in chlorides.

" It has also been proposed to shift the equilibrium in a 2,949,406Patented Aug. 16, 1960 The present invention depends for itseffectiveness inv producing high yields of tetracycline in afermentation medium in which it is not necessary to remove chloride ionsfrom the system. The present invention is based upon the discovery thatwhen a chlorination inhibitor as hereinafter described is added to achloride containing fermentation, the activities of the organism aredirected from the synthesis of chlortetracycline to the synthesis oftetracycline. The invention is of extreme practical importance becauseit obviates the expense and disadvantages of removing chloride ions fromthe fermentation medium. Thus, in accordance with the present inventiona chloride-containing, chlortetracycline fermentation can be easilyconverted to a tetracycline fermentation by the addition to the nutrientmedium of a relatively small amount of a chlorination inhibitor ashereinafter described.

The present invention is not particularly concerned with any specificmicroorganisms except to the extent that it is concerned with those thatproduce both chlortetracycline and tetracycline by fermentativebiosynthesis. Insofar as we are presently aware all such microorganismsare of the genus Streptomyces. The species Streptomyces aureofaciens,first isolated and described by Dr. Duggar U.S. Patent 2,482,055,produces chlortetracycline in fermentation media in which chloride ionsare present. This species, and its numerous natural and induced mutants,will also produce tetracycline when deprived of chloride ions. A numberof other tetracycline producing microorganisms have been mentioned inthe patent literature as alleged distinct species and among these may bementioned S. viridifaciens, S. sayamaensis, S. feofaciens, BL 567201,and still others. The published morphological data on thesemicroorganisms is insufficient to conclusively determine whether or notthey are new species or merely strains of S. aureofaciens. Regardless ofthis, however, the invention is not predicated upon the selection of aparticular species but, as indicated above, of controlling and reducingthe proportion of chlortetracycline that is produced by thesemicroorganisms in fermentation media containing chloride ions.

The various substances that may be employed in accordance with theprocess of the present invention to inhibit the formation ofchlortetracycline, yet permit the production of tetracycline during thefermentation are thioureas which may be represented by the followinggeneral formula in which R is hydrogen or alkyl, and R is hydrogen,alkyl or other substituent such as will be indicated by the structure ofthe following compounds which have been found to be effective ininhibiting the formation of chlortetracycline in a chloride containingfermentation media with strains of Streptomyces aureofaciens. Theseinhibitors of fermentative chlorination include:

Thiourea Dithiobiuret N,N -diethylthiourea N,N -bisthiocarbamylhydrazineN-ethylaminothiourea Thiosemicarbazide The conditions of thefermentation are generally the same as for presently known methods ofproducing tetracycline or chlortetracycline by fermentation. That is,the fermentation medium contains the usual nutrients and mineralsubstances. Suitable nutrient substances which may provide thosenecessary substances include starch, dextrose, cane sugar, glucose,molasses, soybean meal, peanut meal, yeast, meat extracts, peptone,ammonium sulfate, urea, corn steep liquor, distillers solubles, fishmeal and other conventional substances. The inorganic salts includesuchthings as calcium carbonate, ammonium sulfate, ammonium. chloride,sodium dihydrogen phosphate, and the various trace elements such asmanganese, cobalt, zinc, copper, iron, and the like.

The other general conditions of the fermentation, such as hydrogen ionconcentration, temperature, time, rate of aeration, preparation of theinoculum, sterilization, inoculation and the like are conventional andmay be similar to those shown in the U.S. patent to Duggar, 2,482,055,and other subsequent patents which describe the production ofchlortetracycline and tetracycline.

Similarly, the recovery of the tetracycline from the fermentation liquoris conventional and need not be described, as numerous methods ofrecovering tetracycline fromfermentation liquors have been published.

As in the case of the use of bromide ions in depressing fermentativechlorination the inhibitors of the present invention may be somewhattoxic to the fermentation, especially when used in high concentrations.Fortunately, very small amounts of the inhibitors of this inventiondepress the formation of chlortetracycline very markedly. As little asone part per million shows some effect. Ordinarily the preferred rangeswill be from about five parts per million to 500 parts per million.Generally speaking the more inhibitor that is added, the lesschiortetracycline will be produced. On the other hand as continuedamounts of the inhibitor are added the toxic effects begin to exertthemselves and the total yield of antibiotic is reduced. These effectswill be apparent from examples which follow.

A further important aspect of the invention involves the use of theinhibitors described above with bromide ionsin preventing the formationof chlortetracyclines in a chloride-rich medium. In this way it ispossible to almost completely eliminate the toxic effects of both thebromide ion and the inhibitors of the present invention, in many cases.This effect will also be apparent from the examples which follow.Besides reducing toxic eflects, it is possible to obtain very goodresults with much less bromide ion than would be necessary without usingit in conjunction with the inhibitors of the present invention.Accordingly the bromide ion may be used in amounts ranging from aslittle as 0.005 to about 2% by weight preferably in the order of 0.02 to1%.

In order that the invention may be described in greater particularity afew examples will be given to illustrate some of the more importantaspects of it. It will be understood, of course, that these are merelyexamples of some preferred inhibitors and are intended to illustratesome of effects that may be obtained under varying conditions. Manyother inhibitors of the kind named above may be used with similarresults and the invention is not to be construed as beinglimited tothese examples.

, EXAMPLE 1 Fifty ml. of culture medium (pH 7.2) containing 2.8% ofstarch, 0.2% of. molasses, 3% of defatted powdered peanuts, 0.5% of dryyeast, 0.4% of calcium carbonate, 0.8% of sodium bromide, 0.0007% ofMnSO .7H O and 0.003% of ZnSO .7H O was put in culture flasks (500capacity) and, after addition of thiourea as shown in the table,sterilized by heating at 120 C. for five minutes under increasedpressure. After cooling the medium in each flask was inoculated with 1ml. of a seed culture of Streptomyces sayamaensis, and subjected toaerobic fermentation with shaking (frequency, 146 per min; amplitude,cm.) for 72 hours. After this time the culture broth in each flask is atpH 6.37.8 and shows maximum antibiotic. activity. The tetracyclineproduced therein is then determined with the following results:

Table I gamma/ml. Addition Compound level,

p.p.m. Total Percent Tetra- Tetracycline cyeline Control (without sodiumbromide). 0 2, 200 5-10 Control (with sodium bromide) 1, 890 80 Thiourea80 1, 390 98 EXAMPLE 2.

A fermentation medium composed of the following substances was prepared?Corn flour -grams per liter..- 14.5 Starch 47 Corn steep liquor do 25CaCO .do 9 (NH S O -..do 5.6 CoCl .6H O milligrams per liter" 5 MnSO(70%) do.... 80 Lard oil percent by volume 3 Ammonium chloride grams perliter 1.7

To portions of this medium were added varying amounts The mediaweredispensed in of thiosemicarbazide. appropriate amounts into flasks,sterilized, inoculated with vegetable inoculum of S. aureofaciens(strain S77) and incubated at 265 C. on a rotary shaker for 96- Theywere then. assayed for their chlortetra-- cycline and tetracyclinecontents. The results are prehours.

sented in the table below.

Many other organic compounds having the general formula described. aboveand including those specifically named give similar results to thoseshown in the above examples. of these inhibitors are more effective thanothers. the relative proportions of chlortetraeycline and tetracyclineat the end of the fermentation will depend upon a number of factorsincluding the strain of microorganism that is usedas the fermentingagent. Apparently some strains produce-less chlortetracycline thanothers with a given inhibitor. Also theamount of chlo ride ion. in thefermentation medium will affect the ratios of the antibiotics. Obviouslythe less chloride ion in the medium the less chlortetraeycline can beproduced. The medium used in Examples2'to 6 was a chloride richmediumand as expected it was capable of producing.

large amounts of chlortetracycline. Normally in practicing the inventionthe chloride ion content of the medium' should be kept as low aspossible commensurate with the obtaining of a high yield oftetracycline. Low chloride ion' media have not yet been found favorablefor the production of high yields of antibiotic and-the inhibitors ofthe present invention are particularly usefulin cases where thefermentation medium contains more than about ten parts per million ofchloride ions.

We claim:

1. In a method of producing tetracycline with microorganisms of thegenus Streptomyces which produce chlortetracycline in a chloridecontaining fermentation medium the improvement which comprises the stepof It will be understood, of course, that some- Also adding to saidfermentation medium a small amount of a compound having a thioureanucleus.

2. In a method of preparing tetracycline by aerobic fermentation of achloride containing fermentation medium with strains of S. aureofaciensthe improvement which comprises the step of adding from about to 500parts by weight of thiourea.

3. In a method of preparing tetracycline by aerobic fermentation of achloride containing fermentation medium with strains of S. aureofaciensthe improvement which comprises the step of adding from about 5 to 500parts by Weight of dithiobiuret.

4. In a method of preparing tetracycline by aerobic fermentation of achloride containing fermentation medium with strains of S. aureofaciensthe improvement which comprises the step of adding from about 5 to 500parts by Weight of N,N -diethylthiourea.

5. In a method of preparing tetracycline by aerobic fermentation of achloride containing fermentation medium with strains of S. aureofaciensthe improvement which comprises the step of adding from about 5 to 500parts by weight of N,N -bisthiocarbamylhydrazine.

6. In a method of preparing tetracycline by aerobic fermentation of achloride containing fermentation medium with strains of S. aureofaciensthe improvement which comprises the step of adding from about 5 to 500parts by weight of N-ethylaminothiourea.

7. In a method of preparing tetracycline by aerobic fermentation of achloride containing fermentation medium with strains of S. aureofaciensthe improvement 6 which comprises the step of adding from about 5 to 500parts by weight of thiosemicarbazide.

8. In a method of preparing tetracycline with microorganisms of thegenus Streptomyces which tend to produce chlortetracycline in a chloridecontaining medium the improvement which comprises adding to the aqueousfermentation medium a small amount of a compound of the group consistingof thiourea, dithiobiuret, N,N -diethylthiourea, N,N-bisthiocarbamylhydrazine, N-ethylaminothiourea, thiosemicarbazide andfrom about 0.005% to about 2.0% by weight of bromide ions.

References Cited in the file of this patent UNITED STATES PATENTS2,712,517 Gourevitch et a1 July 5, 1955 2,734,018 Minieri et al Feb. 7,1956 2,739,924 Lein et a1. Mar. 27, 1956 2,763,591 Hatch et a1 Sept. 18,1956 FOREIGN PATENTS 316,291 Switzerland Nov. 15, 1956 OTHER REFERENCESMartell et a1.: Chemistry of the Metal Chelate Compounds, PrenticeHall,Inc., 1952, pp. 471, 506.

Gourevitch et a1.: Antibiotics and Chemotherapy, vol. V, No. 8, August1955, pp. 448-452.

Sekizawa: Jour. of Biochemistry, vol. 42, No. 2, pp. 217-219.

NY. Acad. Sci, vol. 60, Art. I, pp. 71-101.

1. IN A METHOD OF PRODUCING TETRACYCLINE WITH MICROORGANISMS OF THE GENUS STRPTOMYCES WHICH PRODUCE CHLORTETRACYCLINE IN A CHLORIDE CONTAINING FERMENTATION MEDIUM THE IMPROVEMENT WHICH COMPRISES THE STEP OF ADDING TO SAID FERMENTATION MEDIUM A SMALL AMOUNT OF A COMPOUND HAVING THIOUREA NUCLEUS. 